Multiplex detection of intracellular or surface molecular targets in single cells

Abstract

This disclosure demonstrates an approach that translates synthetic DNA codes to spatial codes registered in nanoliter microchambers for multiplexed measurement of nearly any type of molecular targets (e.g., miRNAs, mRNAs, intracellular and surface proteins) in single cells.

What is claimed is: 
     
       1. A method comprising:
 (a) contacting cells that comprise nucleic acid targets with at least two conjugates, each conjugate comprising (i) a nucleic acid bait strand linked to (ii) a nucleic acid reporter strand through (iii) a cleavable linker, wherein the nucleic acid bait strand contains a nucleotide sequence complementary to a nucleic acid target of interest, to produce modified cells comprising target-conjugate complexes; 
 (b) loading modified cells of step (a) on a base substrate containing microwells to produce a loaded base substrate, and 
 (c) contacting the loaded base substrate with a cover substrate comprising at least two sets of at least two immobilized nucleic acid capture probes to produce at least two enclosed microwells,
 wherein (i) each of the capture probes of step (c) comprises a nucleotide sequence complementary to a nucleotide sequence of a nucleic acid reporter strand of step (a), (ii) each of the at least two enclosed microwells comprises a set of at least two different capture probes of the cover substrate, and (iii) each capture probe of the set of at least two different capture probes of the cover substrate corresponds to a different nucleic acid reporter strand associated with a different nucleic acid target of interest in the cells of step (a). 
 
 
     
     
       2. The method of  claim 1 , further comprising (d) cleaving the cleavable linkers of the conjugates to release the nucleic acid reporter strands. 
     
     
       3. The method of  claim 1 , further comprising maintaining the enclosed microwells under nucleic acid hybridization conditions to produce reporter-capture complexes immobilized on the cover substrate. 
     
     
       4. The method of  claim 3 , further comprising dissociating the cover substrate containing the immobilized reporter-capture complexes from the base substrate and visualizing at least one reporter-capture complex immobilized on the cover substrate. 
     
     
       5. The method of  claim 4 , further comprising identifying at least one nucleic acid target of interest of step (a). 
     
     
       6. The method of  claim 1 , wherein step (a) comprises contacting cells that comprise nucleic acid targets with at least three conjugates. 
     
     
       7. The method of  claim 6 , wherein step (a) comprises contacting cells that comprise nucleic acid targets with at least five conjugates. 
     
     
       8. The method of  claim 7 , wherein step (a) comprises contacting cells that comprise nucleic acid targets with at least ten conjugates. 
     
     
       9. The method of  claim 1 , wherein the cells are permeabilized or fixed. 
     
     
       10. The method of  claim 1 , wherein the nucleic acid bait strand contains a nucleotide sequence complementary to a ribonucleic acid (RNA) target. 
     
     
       11. The method of  claim 10 , wherein the RNA target is selected from mRNA targets, tRNA targets, rRNA targets and miRNA targets. 
     
     
       12. The method of  claim 1 , wherein the nucleic acid bait strand contains a nucleotide sequence complementary to a deoxyribonucleic acid (DNA) target. 
     
     
       13. The method of  claim 1 , wherein the cleavable linker is selected from photocleavable linkers and enzyme-cleavable linkers. 
     
     
       14. The method of  claim 1 , wherein the nucleic acid reporter strand is linked to a detectable label. 
     
     
       15. The method of  claim 1 , wherein the base substrate is a microchip. 
     
     
       16. The method of  claim 1 , wherein each microwell has a volume of 1-999 nanoliters. 
     
     
       17. The method of  claim 1 , wherein the cover substrate forms a seal with the base substrate to prevent fluid communication among the enclosed microwells. 
     
     
       18. A device comprising:
 (a) a base substrate comprising microwells, each microwell containing at least one cell containing at least two target-conjugate complexes, each complex comprising a nucleic acid target bound to a nucleic acid bait strand linked to a nucleic acid reporter strand through a cleavable linker; and 
 (b) a cover substrate comprising at least two sets of at least two immobilized nucleic acid capture probes, each capture probe comprising a nucleotide sequence complementary to a nucleotide sequence of a nucleic acid reporter strand of (a), wherein the cover substrate is in contact with the base substrate to form at least two enclosed microwells, each of the at least two enclosed microwells containing a set of at least two different capture probes of the cover substrate, and each capture probe of the set of at least two different capture probes corresponds to a different nucleic acid reporter strand associated with a different cellular target. 
 
     
     
       19. The method of  claim 1 , wherein the at least two sets of at least two immobilized nucleic acid capture probes are patterned on the cover substrate. 
     
     
       20. The method of  claim 19 , wherein each of the enclosed microwells comprises at least one patterned set of the at least two immobilized nucleic acid capture probes.